type-A α-toxin (phospholipase C) was purified on a preparative scale by a simple, rapid, two-stage procedure involving precipitation of culture-supernatant fluids with ammonium sulphate at 35 to 50% saturation, followed by isoelectric focusing in a 4·6 gradient. Milligram yields of highly purified α-toxin with 10 to 15% recovery of activity were obtained in single fractions.

Two forms of α-toxin were identified by electrofocusing. The major peak of activity possessed a of 5·49±0·06(α) and the minor, a of 5·25(α). The former was free from detectable collagenase 4·54), hyaluronidase 4·73), θ-toxin 6·56) and neuraminidase. It gave a single precipitin arc on immunoelectrophoresis, but analysis by SDS polyacrylamide disk-gel electrophoresis at high protein-loading revealed a major protein component of molecular weight 53,800 and two minor protein bands. Atomic emission spectro-scopy did not detect the presence of zinc in such preparations. The latter showed a single line of identity with α in Ouchterlony gel diffusion tests and contained a protein component with the same molecular weight as that of α.

Fractions α and α both possessed hot-cold haemolytic, phospholipase-C, and lethal activities. Both hydrolysed lecithin and sphingomyelin. Electro-focusing of α-toxin in the presence of 6m urea resulted in the detection of only one component, α, with a identical to α. It also possessed all three biological activities of α-toxin. Removal of the urea and refocusing was accompanied by the reappearance of α. The occurrence and formation of α could not be interpreted in terms of artefactual causes of multiple forms of proteins identified by isoelectric focusing. These studies provided evidence favouring the suggestion that α and α could be related as conformers, although aggregation or polymerisation could not be entirely excluded as possible alternative explanations.


Article metrics loading...

Loading full text...

Full text loading...


Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error